The overall aim of the studies proposed is to develop a comprehensive and quantitative understanding of the factors that affect the apparent diffusion rate of water and small metabolites in tissues, as measured using nuclear magnetic resonance. An improved model of water diffusion is needed in order to interpret the variations in apparent diffusion in normal tissues and pathological conditions which give rise to contrast in diffusion-weighted magnetic resonance imaging (DWI). DWI is useful for detecting ischemia and evaluating stroke, as well as for characterizing the state and response of tumors. We aim to clarify some specific controversies arising from previous studies of water diffusion, and to develop new insights into the structural features and physiological processes within tissues that affect apparent diffusion rates. In the next funding period we will further develop new methods to measure diffusion over much shorter times and finer spatial scales using so-called temporal diffusion spectroscopy, and will then apply these methods to derive new information on tissue structure and water compartments. We will also extend our studies to non-neural tissues and model systems. To achieve these aims we will implement oscillating gradient spin echo (OGSE) measurements of diffusion of water at 9.4T to probe tissue structure on a scale << 1 micron, and will record OGSE diffusion spectra over the range 0-100 kHz in tissues and tissue models. From these data we will extract the pore (cell) size, intrinsic water diffusion rates and surface to volume ratio of spaces within tissue. We will use the OGSE dispersion data and model fits to establish which of these parameters change as a result of various physiological perturbations. In addition, we will confirm the measurements and predictions from OGSE studies by performing elaborate computer simulations of water in realistic compartmental tissues and by other measurements and histology. We aim to perform measurements in a selection of carefully controlled samples (isolated rat optic nerve; perfused glial cell pellets; in synchronized HeLa cells at different phases of mitosis; in vitro in a perfused muscle preparation; and in mouse tissues in vivo and post-mortem) to extract new indices of tissue structure and the relationship of water diffusion behavior to basic tissue biophysical properties.